Insulin-dependent diabetes mellitus is an autoimmune disease that is characterized by selective destruction of insulin secreting B-cells found in pancreatic islets of Langerhans. Viral infection is one precipitating event that has been proposed to initiate B-cell damage during the development of autoimmune diabetes. Macrophages and macrophage-derived soluble mediators have been shown to participate in viral-induced diabetes in animal models; however, the mechanisms of viral-induced macrophage activation and B-cell death are unknown. The broad goal of this research is to elucidate the biochemical mechanisms of macrophage activation and B-cell death in response to a viral infection. The replicative intermediate double-stranded (ds) RNA, is one component of viral infections that activate the antiviral response in infected cells. We have shown that dsRNA, in combination with interferon (IFN)-y, stimulates macrophage interleukin (IL)- 1 release and nitric oxide production, and induces B-cell damage in an IL-land nitric oxide-dependent manner. This proposal focuses on determining the mechanisms by which a viral infection or dsRNA activate the antiviral response in macrophages and induce B-cell death. There are two specific aims: 1. To test the hypothesis that calcium-independent phospholipase A2 (iPLA2) and extracellular signal-regulated protein kinase (ERK) mediate dsRNA- and virus-induced macrophage activation as predicted by our preliminary data. Proposed experiments will examine: a) the role of IPLA2 in the regulation of inducible nitric oxide synthase (iNOS) expression in response to dsRNA; b) the role of ERK in the regulation of dsRNA-induced IL-i expression and release by macrophages; and c) the potential regulation of iPLA2 and ERK by the dsRNA-dependent protein kinase PKR. 2. To elucidate the cellular signaling mechanisms by which viral infection or dsRNA induce B-cell destruction. Proposed experiments will examine: a) the role of PKR in dsRNA-induced IL-1 expression by pancreatic B-cells; b) the role of the interleukin lB converting enzyme (ICE) in dsRNA- and virus infection-induced iNOS expression by B-cells; and, c) the mechanisms by which dsRNA and viral infection stimulates B-cell apoptosis. A number of biochemical, molecular biological, immunological, histochemical, and transgenic techniques will be utilized to investigate the cellular pathways through which viral infection or dsRNA stimulates macrophage activation and induces islet destruction. It is hoped that insights into the molecular mechanisms of viral mediated B-cell damage gained from the proposed studies will influence the design of novel therapeutic strategies aimed at the prevention of this debilitating disease.